In recent years, with the spread of therapy using molecular target drugs based mainly on antibody drugs, the necessity of quantitating biological substances (for example, antigens) in the cell of the observation target has been increasing for sore efficient design of the molecular target drugs. For confirming the presence of a biological substance, a method of organization analysis is known on the basis of the binding of a biological substance and the fluorescent substance bonded with biological substance recognition site which bind the biological substance (for example, antibodies).
For example, according to the technique described in Patent Document 1, the expression level of a biological substance on the cell membrane is evaluated by staining cell membrane with a certain kind of fluorescent substance, staining a biological substance with another kind of fluorescent substance, and counting the number of fluorescent bright points which reflect the fluorescent substance on the cell membrane (see paragraphs 0012 to 0013, 0060 to 0066, 0107, etc.). However, the technique including a special step of staining the cell membrane as in Patent Document 1 takes labor, compared to the common method of organization analysis in which hematoxylin staining (H staining) or hematoxylin and eosin staining (HE staining) of a tissue slice is performed.
In contrast, according to the technique described in Patent Document 2, the expression level of a biological substance is evaluated on the basis of HE staining of a tissue slice (see paragraphs 0019, 0029, 0085, etc.).
Specifically, according to the technique described in Patent Document 2, a bright field image of a tissue slice processed for HE staining is acquired, a cell nucleus region is extracted from the bright field image, a region of a prescribed distance from a centroid of the extracted cell nucleus region is estimated as a cell region, and the expression level of a biological substance in the cell region is determined on the basis of the cell nucleus and the number of fluorescent bright points includes within the cell region (see paragraphs 0092 to 0104, FIG. 20A, paragraph 0128, etc.).